1. Field of the Invention
The present invention relates to conjugates of the urokinase plasminogen activator (uPA) and its fragments that bind to the cell surface receptor uPA (uPAR), the peptides and constructs labeled to deliver a diagnostic probe or a therapeutic agent to the surfaces of cells expressing uPAR. The proteins and peptides of the invention are capable of carrying a suitable detectable or imageable label that will allow qualitative detection and also quantitation of uPAR levels in vitro and in vivo. Such labeled peptide compositions are therefore useful as diagnostic, prognostic and imaging tools in all diseases and conditions where this receptor plays a pathological or otherwise undesirable role. cDNA probes that detect uPA and uPAR-expressing cells may also be conjugated with similar diagnostic labels and used in in situ hybridization assays. Furthermore, by targeting therapeutic agents that “label” the peptide to uPAR, it is possible to achieve a number of biological effects that include cell death, the inhibition of cell movement and migration and the inhibition of angiogenesis.
2. Description of the Background Art
Urokinase-type plasminogen activator (uPA) has been identified as the initiator of several cascades related to tumor progression, invasion and angiogenesis. The uPA system is strongly linked to pathological processes, such as cell invasion and metastasis in cancer (Danø et al., Adv. Cancer Res., 44:139-266 (1985)).
Cells produce uPA in an inactive form as a 411 amino acid protein, pro-urokinase (pro-uPA) or single-chain uPA (scuPA), which then binds to its receptor, uPAR. This binding event is a prerequisite for the efficient activation of scuPA to two-chain uPA (tcuPA) in a cellular milieu (Ellis et al., J. Biol. Chem., 264:2185-88 (1989)). Pro-uPA is activated by a single proteolytic cleavage between amino acid 158 (Lys) and 159 (Ile) to activate the proenzyme. Cleavage results in the formation of the two-chain active uPA (tcuPA). Cleavage of pro-uPA at the activation site in fact results in a conformational change and in the gain of plasminogen activator activity both with natural and synthetic substrates.
uPA is a three-domain protein comprising (1) an N-terminal “growth factor domain” (GFD), (2) a kringle domain, and (3) a C-terminal serine protease domain. uPAR, the receptor for pro-uPA, is also a multi-domain protein anchored by a glycosylphosphatidyl-inositol anchor to the outer leaf of the cell membrane (Behrendt et al., Biol. Chem. Hoppe-Seyler, 376:269-279 (1995)).
The amino acid sequence of the N-terminus of human pro-uPA (residues 1-44, SEQ ID NO:1) is
Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly1                                    10Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn             20                                      30Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile                             40The structure of pro-uPA is shown in FIG. 1.
Therapeutic use of uPA, pro-uPA, tissue plasminogen activator (tPA) or streptokinase (for thromboembolism) requires very high dosage due in part to their very rapid clearance. Possible reasons for the short half-life include binding to specific circulating inhibitors, binding to receptors, internalization and degradation of inhibitor-bound and/or receptor-bound PA.
uPAR is not normally expressed at detectable levels on quiescent cells and must therefore be upregulated before it can initiate the activities of the uPA system. uPAR expression is stimulated in vitro by differentiating agents such as phorbol esters (Lund et al., J. Biol. Chem. 266:5177-5181 (1991)), by the transformation of epithelial cells, and by various growth factors and cytokines such as VEGF, bFGF, HGF, IL-1, TNFα in endothelial cells and by GM-CSF in macrophages (Mignatti et al., J. Cell Biol. 113:1193-1201 (1991); Mandriota et al., J. Biol. Chem. 270:9709-9716; Yoshida et al., Inflammation 20:319-326 (1996)). This upregulation has the functional consequence of increasing cell motility, invasion, and adhesion (Mandriota et al., supra). More importantly, uPAR appears to be up-regulated in vivo in most human carcinomas examined to date, specifically, in the tumor cells themselves, in tumor-associated endothelial cells undergoing angiogenesis and in macrophages (Pyke et al., Cancer Res. 53:1911-15 (1993) which may participate in the induction of tumor angiogenesis (Lewis et al., J. Leukoc. Biol. 57:747-751 (1995)). uPAR expression in cancer patients is present in advanced disease and has been correlated with a poor prognosis in numerous human carcinomas (Hofmann et al., Cancer 78:487-92 (1996); Heiss et al., Nature Med. 1:1035-39 (1995). Moreover, uPAR is not expressed uniformly throughout a tumor but tends to be associated with the invasive margin and is considered to represent a phenotypic marker of metastasis in human gastric cancer. The fact that uPAR expression is up-regulated only in pathological states involving extracellular matrix remodeling and cell motility such as cancer makes it an attractive marker for diagnosis as well as a selective target for therapy.
Earlier studies with peptide fragments within the GFD of uPA had shown that residues 20-30 conferred the specificity of binding, but that residues 13-19 were also needed if residues 20-30 were to attain the proper binding conformation. Specifically, the peptide [Ala19]uPA(12-32), which contains two cysteines (the third cysteine being replaced by Ala to avoid undesired disulfide bond formations) in its open chain form prevented uPA binding to uPAR with an IC50 of 100 nM. In its oxidized cyclic form with an intrachain disulfide bond between Cys13 and Cys31, the peptide prevented uPA binding with an IC50 of 40 nM. The authors proposed that residues 13-19 might act indirectly to provide a scaffold that would help residues 20-30 attain the correct binding conformation (Appella et al., J. Biol. Chem., 262:4437-4440 (1987).
A related commonly-assigned patent (U.S. Pat. No. 5,942,492) and application (U.S. Ser. No. 09/1,816), both of which are incorporated herein by reference in their entirety, show that novel cyclic molecules derived from the uPA peptide fragment 20-30 (in which residue 20 is covalently bonded to residue 30) bind to uPAR with IC50 values in the 10-100 nM range.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.